Microbial Production of Cellulosic Isobutanol: Integrating Ecology and Evolutionary Approaches with Engineering.

Biofuels derived from lignocellulosic feedstocks are widely considered to be among the most promising renewable fuels that can be produced at a large scale and in a sustainable manner. However, many challenges exist. In this work, we aim to address two of them, which are interconnected under an overall goal of achieving efficient microbial conversion of lignocellulosic feedstocks to isobutanol, an advanced biofuel: i) enabling consolidated bioprocessing of lignocellulosic feedstocks to biofuels, through engineering synthetic microbial consortia; and ii) improving microbial stress tolerance, through genome evolution and engineering. Inspired by the versatility and robustness of ubiquitous natural microbial ecosystems, the first part of our work explores engineering synthetic multispecies microbial communities for cellulosic biofuel production. The required biochemical functions are divided between two specialist organisms: the fungus Trichoderma reesei, which secretes cellulases to hydrolyze lignocellulose into soluble saccharides, and the bacterium Escherichia coli, which metabolizes soluble saccharides into isobutanol. We developed and experimentally validated a comprehensive modeling framework, allowing us to elucidate key ecological interactions and develop mechanisms for stabilizing and tuning population composition. To illustrate bioprocessing applications, we demonstrate direct conversion of cellulosic feedstocks to isobutanol, achieving titers up to 1.86 g/L and 62% of theoretical yield. In the second part, we leverage recent advances in DNA sequencing and genome engineering technologies to decode and refactor microbial tolerance to isobutanol, a complex phenotype with a poorly understood genetic basis. We experimentally evolved isobutanol tolerant E. coli strains, and then used genome re-sequencing and functional dissection studies to reverse engineer mechanisms and genetic bases of tolerance. Next, we exploited our initial results to select genetic loci for targeted mutagenesis using Multiplex Automated Genome Engineering (MAGE), allowing us to refactor isobutanol tolerance and explore large genotype spaces for hyper-tolerant variants. In summary, we have integrated ecology and evolutionary approaches with engineering to develop novel microbial systems for biofuel production. Our synthetic microbial consortium approach provides key advantages over the conventional paradigm of engineering a single microbe (“super-bug”); in parallel, our genome evolution and engineering work has generated new insights into genetic and biochemical mechanisms underlying microbial tolerance to toxic chemicals.PhDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99954/1/jminty_1.pd